Development of a new translation system for animal proteins: the engineering strategy of ribosome function

在本项目中，我们分析了翻译机制中的功能结构，并利用分子工程对其进行了修饰，以开发一种新的翻译系统，用于真核蛋白质的合成。以下是该项目的主要成果1.位于核糖体GTPase相关中心的蛋白质复合体的功能结构。在真核生物中，一对P1-P2异源二聚体结合到PO的C-末端区域，形成五聚体复合体。在真细菌中，2~3个L12同源二聚体(不同种类)结合在L10的C末端。生化分析和X射线结晶学分析表明，在古细菌中，有3个L12同源二聚体与PO结合并形成七聚体复合体。通过从每个复合体中去除一个或两个二聚体，降低了GTP酶的活性和多肽的合成。昆虫病毒IRES介导的翻译启动机制：1)IRES通过与小亚基蛋白S25相互作用与核糖体结合；2)诱导核糖体发生构象变化；3)激活核糖体对延伸因子eEF2的可及性；4)增强第一个氨酰tRNA与核糖体P位点的结合。我们还利用IRES-荧光素酶融合mRNA成功地翻译了IRES介导的荧光素酶。利用该系统，我们明确了携带PSIV IRES基因的3‘-非翻译区参与了翻译效率。一种新的体内翻译系统的开发。大肠杆菌突变株DL11缺乏L11核糖体蛋白，L11核糖体蛋白是GTPase相关中心的组成部分。该菌株生长速度很低(野生型114~115)，是蛋白质合成的宿主细胞。我们证明了该菌株对于合成普通菌株不能以可溶状态获得的真核细胞蛋白是有用的。

In this project, we analyzed functional structures in the translational machinery and modified them by molecular engineering in order to develop a new translation system useful for synthesis of eukaryotic proteins. The following are major results of this project.1. The functional structure of protein complex located in the ribosomal GTPase-associated center.In eukaryotes, a couple of P1-P2 heterodimers bound to the C-terminal regions of PO and formed a pentameric complex. In eubacteria, 2〜3 L12 homodimers (differing with species) bound to the C-terminus of L10. In archaebacteria, 3 L12 homodimers bound to PO and formed a heptameric complex, as revealed by biochemical analysis and X-ray crystallography. By removing one or two dimers from each the complex, the activity of GTPase and polypeptide synthesis was reduced.2. Mechanism of translation initiation mediated with the IRES of an insect virus (PSIV).We elucidated that 1) the IRES binds to the ribosome through interaction of the small subunit protein S25, 2) induces some conformational changes in the ribosome, 3) activates the ribosomal accessibility to elongation factor eEF2, and then 4) enhances binding of the first aminoacyl-tRNA to the ribosomal P site. We also succeeded translation of luciferase mediated by the IRES using an IRES-luciferase fusion mRNA. By using this system, we clarified that the 3'-untranlated region of the PSIV IRES-carrying gene participates in the translation efficiency.3. Development of a new in vivo translation system.The E.coli mutant strain dL11 lacks L11 ribosomal protein that is a component of the GTPase-associated center. This strain showed very low growing rate (114〜115 of wild type) and was used for a host cell of protein synthesis. We demonstrated that this E. coli strain is useful for synthesis of eukatyotic proteins that cannot be obtained as a soluble state by usual strains.